American Society of Hematology Self-Assessment Program

772 Pages·2019·18.977 MB·English

by  Adam Cuker, Jessica K. Altman, Aaron T. Gerds, Ted Wun

Table of Contents
Chapter Page
1 Molecular basis of hematology 1
2 Consultative hematology I : hospital- based and selected outpatient topics 26
3 Consultative hematology II : women’s health issues 61
4 Hematopoietic growth factors 96
5 Iron physiology, iron overload, and the porphyrias 115
6 Acquired underproduction anemias 138
7 Thalassemia, sickle cell disease, and other hemoglobinopathies 161
8 Hemolytic anemias excluding hemoglobinopathies 186
9 Thrombosis and thrombophilia 217
10 Bleeding disorders 260
11 Disorders of platelet number and function 291
12 Laboratory hematology 321
13 Transfusion medicine 350
14 Cellular basis of hematopoiesis and stem cell transplantation 386
15 Clinical hematopoietic cell transplantation and adoptive cell therapy 408
16 Inherited marrow failure syndromes and myeloid disorders 454
17 Chronic myeloid leukemia 486
18 Myeloproliferative neoplasms 510
19 Acquired marrow failure syndromes: aplastic anemia, PNH, and MDS 546
20 Acute myeloid leukemia 580
21 Acute lymphoblastic leukemia and lymphoblastic lymphoma 593
22 Hodgkin lymphoma 621
23 Non- Hodgkin lymphoma 651
24 Chronic lymphocytic leukemia/small lymphocytic lymphoma 700
Molecular basis of hematology
1
T A. G O A -W
ANJA RUBER AND MAR BDEL AHAB
Basic concepts 1
Analytic techniques 9
Clinical applications of DNA Basic concepts
technology in hematology 18
Advances in recombinant DNA technology over the past several de cades have
Glossary 22
substantially altered our view of biologic pro cesses and have immediate rele-
Bibliography 25 vance to our understanding of both normal hematopoietic cell function and
hematologic pathology. A complete review of molecular ge ne tics is beyond the
scope of this chapter, but it is intended as a review of the concepts of the mo-
lecular biology of the gene, an introduction to epigenet ics and genomics, an
outline of noncoding RNAs, concepts relevant for immunotherapeutic treat-
ment approaches, and an explanation of the terminology necessary for under-
standing the role of molecular biology in breakthrough discoveries. Emerging
diagnostic and therapeutic approaches in hematology are reviewed. The con-
cepts outlined in the following sections also are illustrated in Figure 1-1; in ad-
dition, boldface terms in the text are summarized in the glossary at the end of
this chapter. Several examples of how these concepts and techniques are applied
The online version of this in clinical practice are included.
chapter contains an educational
multimedia component on normal Anatomy of the gene
hematopoiesis.
Structure of DNA
DNA is a complex, double-s tranded molecule composed of nucleotides. Each
nucleotide consists of a purine (adenine or guanine) or pyrimidine (thymine
or cytosine) base attached to a deoxyribose sugar residue. Each strand of DNA
is a succession of nucleotides linked through phosphodiester bonds between
the 5′ position of the deoxyribose of one nucleotide and the 3′ position of the
sugar moiety of the adjacent nucleotide. The two strands are connected through
hydrogen bonds between strict pairs of purines and pyrimidines; that is, adenine
must be paired with thymine (A- T) and guanine must be paired with cytosine
(G- C). This is known as Watson- Crick base pairing. Consequently, the two
strands of DNA are said to be complementary, in that the sequence of one
Confl ict- of- interest disclosure: strand determines the sequence of the other through the demands of strict base
Dr. Gruber declares no competing
pairing. The two strands are joined in an antiparallel manner so that the 5′ end
fi nancial interest. Dr. Abdel- Wahab
of one strand is joined with the 3′ end of the complementary strand. The strand
declares no competing fi nancial interest.
containing the codons for amino acid sequences is designated as the sense strand,
Off- label drug use: Dr. Gruber:
whereas the opposite strand that is transcribed into messenger RNA (mRNA) is
not applicable. Dr. Abdel-W ahab:
not applicable. referred to as the antisense strand.
1
2 1. Molecular basis of hematology
Nucleus
Coding Noncoding
sequence (intervening 3’ coding
(exon) sequence, intron) strand
5’
DNA
3’ 5’ noncoding
Transcription
strand
mRNA 5’ Exon Intron
3’
precursor
5’ CAP 3’ Poly (A),
modification and
Processing shortening of
transcript
Processed 5’ CAP Poly (A)-3’
mRNA
transcript
Nuclear “pore” 5’ CAP Poly (A)-3’ Cytoplasm
mRNA
Transport to
cytoplasm
Initiation factors,
Translation tRNA, ribosomes
5’ CAP Poly (A)-3’
Completed apoprotein
Cofactors and other subunits
Microsomes, Golgi, etc.
Completed functioning protein
Figure 1-1 Flow of ge ne tic information from DNA to RNA to protein. DNA is shown as a double-s tranded array of
alternating exons (red) and introns (pink). Transcription, posttranscriptional proc essing by splicing, polyadenylation, and capping are
described in the text. The mature transcript passes from the nucleus to the cytoplasm, where it is translated and further modified to
form a mature protein. Reproduced with permission from Hoffman R et al, eds., Hematology: Basic Princi ples and Practice, 6th ed.
(Philadelphia, PA: Saunders Elsevier, Inc; 2013:5).
trol the timing and the level of its expression, is termed a
Structure of the gene gene. The gene contains several critical components that
DNA dictates the biologic functions of the organism by determine both the amino acid structure of the protein it
the flow of ge ne tic information from DNA to RNA to encodes and the mechanisms by which the production of
protein. The functional gen e tic unit responsible for the pro- that protein may be controlled. The coding sequence,
duction of a given protein, including the ele ments that con- which dictates protein sequence, is contained within
Basic concepts 3
exons; these stretches of DNA may be interrupted by in- nuclear ribonuclear proteins (snRNPs). mRNA splicing
tervening noncoding sequences, or introns. In addition, is an impor tant mechanism for generating diversity in the
t here are flanking sequences in the 5′ and 3′ ends of the proteins produced by a single gene. Some genes exhibit
coding sequences that often contain impor tant regulatory alternative splicing, a pro cess by which certain exons
ele ments that control the expression of the gene. are included in or excluded from the mature mRNA, de-
Genes are arrayed in a linear fashion along chromo- pending on which splice sequences are used in the ex-
somes, which are long DNA structures complexed with cision pro cess. For example, this is the means by which
protein. Within chromosomes, DNA is bound in chro- some erythroid- specific proteins of heme synthesis (ami-
matin, a complex of DNA with histone and non- histone nolevulinic acid synthase) and energy metabolism (pyru-
proteins that “shield” the DNA from the proteins that vate kinase) are generated, contrasting with the alterna-
activate gene expression. The ends of chromosomes are tively proc essed genes in the liver and other tissues. This
capped by complexes of repetitive DNA sequences and as- permits functional diversity of the products of the same
sociated proteins known as telomeres. The DNA replica- gene and is one of several determinants of tissue speci-
tion machinery cannot effectively replicate the very ends ficity of cellular proteins. Mutations in the sequences of
of chromosomes, and thus shortening occurs with each either introns or exons can derange the splicing pro cess by
cell division, ultimately leading to chromosomal instabil- either creating or destroying a splice site so that the intron
ity and cellular senescence. Telomeres are therefore criti- sequence is not removed or the exon sequence eliminated.
cal to protecting chromosome ends from degradation and If abnormal splicing results in a premature stop codon
fusion, and mutations in genes encoding components of (nonsense mutation), then a surveillance pathway known
the telomere complex are associated with the bone mar- as nonsense- mediated decay may result in degrada-
row failure syndrome dyskeratosis congenita. Furthermore, tion of the abnormal mRNA. This mechanism generally
inappropriate activation of the enzymes that maintain telo- applies to stop codon mutations in the first one- third to
meres (“telomerase”) is associated with numerous cancers, one- half of the mRNA and works to prevent synthesis of
including hematologic malignancies. mutant peptides. When mutations occur in the last one-
third of the mRNA molecule, abnormal peptides may be
produced.
Flow of ge ne tic information
Mutations affecting the spliceosome machinery are
Transcription found in a variety of hematopoietic malignancies. For
RNAs are mostly single-s tranded molecules that differ example, splicing factor mutations (SF3B1, U2AF1, and
from DNA in two ways: by a sugar backbone composed SRSF2) are found in approximately 10% of patients with
of ribose rather than deoxyribose, and by containing the chronic lymphocytic leukemia and 50% of patients with
pyrimidine uracil rather than thymine. The first step in myelodysplastic syndrome (MDS), as well as related myeloid
the expression of protein from a gene is the synthesis of malignancies. These mutations have now been shown to
a pre- messenger RNA (pre- mRNA). The transcrip- alter splicing in a manner distinct from loss- of- function, but
tion of pre- mRNA is directed by RNA polymerase II, the connection between these changes in splicing and clonal
which in conjunction with other proteins generates an hematopoietic disorders is not yet well understood.
RNA copy of the DNA sense strand. The introns are then
removed by a complex pro cess called mRNA splicing. Translation
This pro cess involves the recognition of specific sequences The mature mRNA is transported from the nucleus to the
on either side of the intron which allow its excision in cytoplasm, where it undergoes translation into protein.
a precise manner that maintains the exon sequence. The The mRNA is “read” in a linear fashion by ribosomes,
mRNA may then undergo modifications at the 5′ and which are structures composed of ribonucleoprotein that
3′ ends (capping and polyadenylation, respectively). move along the mRNA and insert the appropriate amino
Although RNA splicing is largely restricted to the nucleus, acids, carried by transfer RNAs (tRNAs), into the na-
it also can occur in the cytoplasm of platelets and neutro- scent protein. The amino acids are encoded by three base
phils activated by external stimuli. triplets called codons, the ge ne tic code. The four bases
Splicing of mRNA is a critical step in gene expression, can encode 64 pos sib le codons; b ecause there are only 20
with impor tant implications for understanding hemato- amino acids used in protein sequences, more than one
logic disease. Splicing is controlled by the spliceosome, codon may encode the same amino acid. For this reason,
a large complex of proteins (100 to 300) and five small the ge ne tic code has been termed degenerate. An amino
4 1. Molecular basis of hematology
acid may be encoded by more than one codon; however, first 50 bases 5′ to the structural gene and is called the
any single codon encodes only one amino acid. The be- promoter region. Other sequences that regulate the level
ginning of the coding sequence in mRNA is encoded by of transcription of the gene are located at less predict-
AUG codon that has variable translation initiation activ- able distances from the structural gene. Such sequences
ity determined by the neighboring nucleotide sequences may increase (enhancers) or decrease (silencers) expres-
(Kozak sequence). In addition, there are three termina- sion. A special type of enhancer is the locus control re-
tion codons (UAA, UAG, and UGA) that signal the end gion, which was first defined in the β- globin cluster of
of the protein sequence. genes on chromosome 11. It is located approximately 50
Single- base- pair alterations in the coding sequence of kilobases (kb) upstream from the β- globin gene, controls
genes (point mutations) may have a range of effects on the all genes within the β- globin locus, and also has a strong
resultant protein. Because the ge ne tic code is degenerate, tissue- specific activity (erythroid- specific).
some single-b ase- pair changes may not alter the amino acid Control of gene expression is exerted through the in-
sequence, or they may change the amino acid sequence in teraction of the cis- acting ele ments described previously
a manner that has no effect on the overall function of the with proteins that bind to t hose sequences. T hese nuclear
protein; these are predicted to be phenotypically silent mu- DNA binding proteins are termed trans- acting factors
tations. In other cases, mutations may lead to a loss or gain or transcription factors. Most of these proteins have a
of protein function, or may result in the acquisition of a DNA- binding domain that can bind directly to regulatory
new function (missense mutation). Sickle cell disease sequences within the gene locus; many of them contain
is an example of a single base-p air change, resulting in an common motifs, such as zinc fin gers or leucine zip-
amino acid alteration that critically changes the chemi- pers, which are shared by many transcription f actors. In
cal characteristics of the globin molecule. Other mutations addition, they frequently have unique domains that allow
may change a codon to a termination codon, resulting in them to interact with other transcription f actors. Thus, a
premature termination of the protein (nonsense muta- complex pattern exists whereby the expression of diff er ent
tion). Fin ally, single or multiple base- pair insertions or dele- transcription factors, which may interact both with one
tions can disrupt the reading frame of genes. These frame- another and with specific regions of DNA to increase or
shift mutations render the gene incapable of encoding decrease transcription, determines the unique tissue and
normal protein. These latter two abnormalities account for stage- specific expression of the genes within a given cell.
some β- thalassemias and for polycythemia due to a gain of
function in the erythropoietin receptor. Clinically impor- Epigenet ics
tant mutations also may occur in the noncoding region of For a gene to be expressed, chromatin must be unwound
genes, such as in the regulatory elem ents upstream of the and the DNA made more accessible to regulatory proteins.
initiation codon or within intronic splicing sites. This is controlled by epige ne tic pro cesses or modifications
to the genome that regulate gene expression without al-
Control of gene expression tering the under lying nucleotide sequence. These changes
With the exception of lymphocytes (which undergo may be modulated by environmental f actors and may be
unique changes in the DNA encoding immunoglobulin or heritable. Epige ne tic modulation of gene expression was
the T- cell receptor) and germ cells (which contain only half first recognized in studies of glucose-6- phosphate dehydro-
of the DNA of somatic cells), each nucleated cell in an in- genase (G6PD), a protein encoded by an X- linked gene.
dividual has the same diploid DNA content. Consequently, Ernest Beutler deduced the princi ple of random embry-
biologic pro cesses are critically dependent on gene regu- onic X chromosome inactivation from studies of G6PD
lation, the control of gene expression such that proteins deficiency. His observations and the studies of Mary Lyon
are produced only at the appropriate time within the ap- and Susumu Ohno on the mechanism of dosage compen-
propriate cells. Gene regulation is the result of a complex sation in mammals led to an understanding of X chromo-
interplay of specific sequences within a gene locus, chro- some inactivation in females. This was the first example
matin, and regulatory proteins (transcription factors) that of stochastic epigen e tic silencing in humans, demonstrat-
interact with those sequences to increase or decrease the ing that human females are mosaics of the activity of X
transcription from that gene. chromosome– encoded genes. Using this princi ple in tu-
DNA sequences that lie in proximity to and regulate mor tissue derived from females led to early demonstrations
the expression of genes, which encode protein, are termed that neoplastic diseases are, for the most part, clonal. Two
cis- acting regulatory ele ments. Nearly all genes have common forms of epige ne tic changes are DNA methyla-
a site for binding RNA polymerase II that is within the tion and histone modifications.
Basic concepts 5
active copies of the Prader-W illi–a ssociated genes and re-
DNA methylation sult in Prader- Willi syndrome.
In addition to being complexed with protein, DNA is As DNA methylation modulates gene activity, acquired
modified by the addition of methyl groups to cytosine changes in methylation patterns are thought to contribute
residues (resulting in 5- methylcytosine) through en- to functional alterations in hematopoietic stem cells dur-
zymes called DNA methyltransferases. Methylation nor- ing aging. Hematopoietic stem cells display an age- related
mally occurs throughout the genome and is associated decline in function and a relative loss of lymphoid differ-
with alterations in gene expression and proc esses such as entiation potential, and these changes are associated with
X- chromosome inactivation, imprinting, aging, and car- site- specific changes in DNA methylation. Furthermore,
cinogenesis. It is generally a marker of an inactive gene, aberrant methylation may contribute to cancer. For ex-
and changes in gene expression often can be correlated ample, mutations in enzymes affecting DNA methylation,
with characteristic changes in the degree of methyla- most notably the DNA methyltransferase DNMT3A, are
tion of the 5′ regulatory sequences of the gene. DNA common in acute myeloid leukemia (AML). Mutations
methylation can silence genes through two mechanisms. in DNMT3A are found in approximately 20% of adult
In the first, the methylation mark itself can impair bind- AML patients and are associated with poor outcomes. T hese
ing of transcription factors to the gene. Second, methylated mutations appear to be acquired early in the evolution of
DNA can be bound by proteins known as methyl- CpG- the disease, thus likely serving as initiators of the disease.
binding domain proteins that recruit additional chroma- Other enzymes affecting DNA methylation, including
tin remodeling proteins to the locus, which then modify TET2 and IDH1/2, are also recurrently mutated in AML,
histones leading to the formation of compact, inactive further illustrating the role of aberrant methylation in the
chromatin termed h eterochromatin. In contrast to 5′ regu- pathogenesis of this disease. Of note, oxidation of methyl-
latory sequences where methylation is typically associated ated cytosines (5- methylcytosine) by members of the ten-
with gene silencing, methylation of intragenic cytosine resi- eleven translocation (TET) protein family results in the
dues often coincides with active transcription of the gene formation of 5- hydroxymethylcytosine, 5-f ormylcytosine,
within which they lie. The function and mechanism of and 5- carboxylcytosine. While the role of these epige ne-
gene body methylation are not well understood. tic marks is not clearly understood, their levels are often
Mendelian ge ne tics is based on the princip le that the altered in cancers (including AML), suggesting that they
phenotype is the same w hether an allele is inherited from may contribute to the pathogenesis of disease. Given the
the mother or the father, but this does not always hold prominent role of abnormal methylation in hematologic
true. Some human genes are transcriptionally active on malignancies, small molecule inhibitors of DNA meth-
only one copy of a chromosome (such as the copy inher- yltransferases (eg, 5-a zacitidine, decitabine) are used in the
ited from the f ather), whereas the other copy on the chro- treatment of disorders that are characterized by aberrant
mosome inherited from the m other is transcriptionally DNA methylation (eg, MDS, AML).
inactive. This mechanism of gene silencing is known as
imprinting, and these transcriptionally silenced genes are Histone modification
said to be “imprinted.” When genes are imprinted, they Histones are DNA packaging proteins that org a nize DNA
are usually heavil y methylated in contrast to the nonim- into structural units called nucleosomes. Octamers of the
printed copy of the allele, which typically is not methyl- core histones— H2A, H2B, H3, and H4—m ake up the
ated. A classic example of imprinting is the inheritance of nucleosome around which 147 bp of DNA is wrapped,
Prader- Willi and Angelman syndromes, which are associ- and histone H1 binds the “linker” DNA between nucleo-
ated with a 4- megabase (Mb) deletion of chromosome 15. somes. Histones are subject to multiple modifications—
This region contains the gene associated with Angelman including methylation, acetylation, ubiquitination, phos-
syndrome, UBE3A, which encodes a ligase essential for phorylation, and others. The par tic u lar combination of
ubiquitin- mediated protein degradation during brain de- histone modifications at any given locus is thought to
velopment. This gene is imprinted on the paternal allele. confer a “histone code,” regulating proc esses such as gene
In addition, the region contains multiple genes associated expression, chromosome condensation, and DNA repair.
with Prader- Willi syndrome, which are imprinted on the Like methylation, histone modifications regulate gene ac-
maternal allele. Thus, maternal inheritance of a mutation tivity, and therefore disruptions of the normal pattern of
or deletion in UBE3A removes the single active copy of these modifications can contribute to cancer and other
the gene and results in Angelman syndrome, and paternal diseases. For example, hypoacetylation of histones H3 and
inheritance of deletions in this region remove the only H4 is associated with silencing of the cell cycle regulator
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6 1. Molecular basis of hematology
p21WAF1, a gene whose expression is reduced in multiple
Nucleus
tumor types. Aberrant expression levels of histone deacet- Cleavage
ylases, the enzymes that remove acetyl groups from his- “Cropping”
tone tails, are common in hematopoietic malignancies, and AAAAA
histone deacetylase inhibitors are being tested in a variety
of these diseases. The first of these inhibitors, vorinostat,
Cytosol
is used in the treatment of cutaneous T-c ell lymphoma. In
Cleavage
addition to altered expression levels of histone- modifying
“Dicing”
enzymes, several fusion genes associated with hematologic
malignancies aberrantly recruit histone modifiers to target
Argonaute and
genes, resulting in altered transcription (eg, the association
other proteins RISC
of MLL fusion proteins with the DOT1L enzyme that 3’ 5’
methylates histones and the histone acetyltransferase p300 Extensive match Less extensive match
with AML1-E TO). Targeting histone modifiers therapeu-
tically, therefore, has potential utility for a variety of cancers. AAAAA AAAAA
mRNA mRNA
“Splicing”
Noncoding RNAs
It has been estimated that only approximately 1% to 2%
AAAAA
of the genome encodes protein, but a much larger fraction ATP
is transcribed. This transcribed RNA that does not en- ADP RISC released
code protein is referred to as noncoding RNA (ncRNA)
and is grouped into an increasingly large number of dif fer- AAAAA Translation reduced
Transfer of mRNA into
ent classes, including microRNAs, small nucleolar RNAs
P-bodies and eventual
(snoRNAs), small interfering RNAs (siRNAs), Piwi- Rapid mRNA degradation degradation
interacting RNAs (piRNAs), long noncoding RNAs
Figure 1-2 MicroRNA production. Production of microRNA
(lncRNAs), and many others. Although each of these begins with transcription of the microRNA gene to produce a
classes of ncRNAs differs in size, biogenesis pathway, and stem- loop structure called a pri- microRNA. This molecule is
specific function, they share a common ability to recognize proc essed by Drosha (“cropping”) to produce the shorter pre-
microRNA. The pre- microRNA is exported from the nucleus; the
target nucleotide sequences through complementarity and
cytoplasmic Dicer enzyme cleaves the pre-m icroRNA (“dicing”)
regulate gene expression. The most well- described class of to produce a double-s tranded mature microRNA. The mature
ncRNAs are microRNAs (miRs), whose biogenesis path- microRNA is transferred to RISC (RNA-i nduced silencing
way is illustrated in Figure 1-2. Following transcription, a complex), where it is unwound by a helicase. Complementary
base pairing between the microRNA and its target mRNA directs
portion of this RNA (the pri- microRNA) forms hairpin
RISC to destroy the mRNA (if completely complementary) or
loops that are cleaved by the enzymes Drosha and Dicer halt translation (if a mismatch exists at the scissile site). Reproduced
into short 21- to 23-bp double-s tranded RNAs. T hese with permission from Alberts B et al, Molecular Biology of the
short double-s tranded RNAs contain both sense strands Cell, 5th ed. (New York, NY: Garland Science; 2007).
and antisense strands that correspond to coding sequences
in mRNAs. T hese mature miRs then are incorporated
into a larger complex known as an RNA-i nduced si- hematopoiesis, numerous miRs that influence cell fate de-
lencing complex (RISC). The miR is then unwound cisions have been identified. miR-223, for example, plays
in a strand-s pecific manner, and the single-s tranded RNA a central role in myeloid differentiation, and reduced ex-
locates mRNA targets by Watson-C rick base pairing. Gene pression of miR-223 is common in AML. Conversely, the
silencing results from cleavage of the target mRNA (if expression of miR-125b, which is expressed in normal
there is complementarity at the scissile site) or transla- hematopoietic stem cells and whose targets include genes
tional inhibition (if there is a mismatch at the scissile site). involved in regulating cell proliferation, differentiation,
This gene-s ilencing pathway is known as RNA interfer- and survival, is increased in a variety of myeloid and lym-
ence. As mediators of gene expression, miRs and other phoid malignancies.
ncRNAs are expressed in a tissue-s pecific manner and play
impor tant regulatory roles in development and differentia- Molecular basis of neoplasia
tion. Accordingly, dysregulation or mutations in ncRNAs Normal cellular growth and differentiation depends on
are associated with vario us diseases, including cancer. In the precise control of gene expression, and alterations in
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Basic concepts 7
the quantity or timing of gene expression can affect the tance of heterozygous mutations in tumor suppressor genes.
survival and function of a cell. When such alterations oc- For example, Li-F raumeni syndrome results from inherited
cur in certain types of genes known as oncogenes or tu- mutations in the cell cycle regulator TP53 and is associated
mor suppressor genes, the cell may gain abnormal growth with the early onset of multiple tumor types; including os-
or survival properties, and accumulations of such muta- teosarcoma, breast cancer, leukemia, and o thers. When mu-
tions may lead to cancer. tations occur in the remaining normal allele, termed “loss
of heterozygosity,” tumor growth is initiated. In some cases,
Oncogenes loss of just one copy of a gene (“haploinsufficiency”) has
Oncogenes are genes that have the potential to cause can- been shown to contribute to cancer development. For
cer, and they arise from mutations in their normal counter- example, loss of one copy of the ribosomal gene RPS14 in
parts termed proto- oncogenes. Proto- oncogenes generally patients with 5q- syndrome leads to aberrant ribosomal
code for proteins or ncRNAs that regulate such pro cesses protein function and a block in erythroid differentiation.
as proliferation and differentiation, and activating muta-
tions or epige ne tic modifications that increase the ex- Neoplasia and the immune system
pression or enhance the function of these genes confers a The immune system defends and protects an individual
growth or survival advantage on a cell. The first described through the detection of “nonself” antigens from either
oncogene, termed SRC, was discovered in the 1970s and is pathogens or infected/malignant cells, followed by ex-
a member of a f amily of tyrosine kinases that regulate cell pansion of effector cells that destroy them, as well as the
proliferation, motility, adhesion, survival, and differentiation. development of immunological memory for subsequent
Activating mutations in the SRC f amily kinases are associ- defense. The ability of cancer to evade or escape the im-
ated with the pathogenesis of multiple types of neoplasias; mune system is a hallmark of the disease and forms the
including cancers of the colon, breast, blood, head and neck, basis of so-c alled immunotherapeutic approaches. T hese
and others. Another classic example of an oncogene is the approaches include increasing the immunogenicity of can-
BCR-A BL1 fusion gene found in chronic myelogenous cer cells, as well as enhancing the immune response to the
leukemia (CML). This fusion results from a translocation cells through a variety of mechanisms—i ncluding admin-
between the BCR gene on chromosome 9 and the ABL1 istration of cytokines, blocking negative regulators of
proto-o ncogene on chromosome 22 and confers constitu- T- cell function, and engineered cellular therapies. Two
tive activation of ABL1 and enhanced cell proliferation. of the most successful approaches to date are immune
Pharmacologic targeting of the activity of oncogenes, such checkpoint inhibitors and chimeric antigen recep-
as the use of the tyrosine kinase inhibitor imatinib to treat tor T (CAR T) cells, both of which enhance T- cell re-
CML, can be an effective therapeutic approach. sponses to tumor cells.
T cells initiate an immune response through the recog-
Tumor suppressors nition of antigenic tumor peptides presented by the major
In contrast to oncogenes, tumor suppressors are genes histocompatibility complex (MHC) protein on the surface
that encode proteins or ncRNAs whose normal function of either antigen- presenting or tumor cells by the T- cell
is to inhibit tumor development through the promotion receptor (TCR). TCR engagement alone is insufficient
of such proc esses as apoptosis, DNA repair, cell cycle inhi- to activate T cells; they require an additional costimulatory
bition, cell adhesion, and o thers. Loss of the expression or signal by the CD80/B7 protein that engages with CD28
function of these genes is associated with cancer, and gen- on T cells. In contrast, engagement of the CTLA-4 protein
erally both copies of the tumor suppressor gene must be expressed on T cells by CD80/B7 leads to cell cycle arrest.
altered to promote neoplasia. Thus, most tumor suppres- This proc ess modulates early steps in T-c ell activation. Dur-
sors follow the “two-h it hypothesis” proposed by Alfred ing long-t erm antigen exposure, T cells upregulate PD-1,
Knudson in his study of the retinoblastoma- associated which inhibits T cells upon interaction with PD-L 1 that is
tumor suppressor gene RB1. This gene encodes a protein expressed on tumor cells. Thus CTLA-4 modulates early
that functions to regulate cell cycling and survival. B ecause T-c ell activation, whereas PD-1 functions in the effector
both copies of the gene must be mutated for retinoblas- phase. Immune checkpoint inhibitors that target both of
toma to manifest, individuals that inherit a mutant allele these stages have been developed. Anti-C TLA-4 mono-
(requiring just one more “hit” in the remaining normal clonal antibodies modulate early steps in T- cell activation,
allele for loss of gene function) generally develop disease whereas monoclonal antibodies directed against PD-1 or
earlier than t hose that must acquire “hits” in both alleles. PD- L1 act to reverse inhibition of T cells that are pre sent
Familial cancer syndromes often result from the inheri- in the tumor microenvironment (Figure 1-3A).
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A Antigen-presenting cell Naive T cell Effector T cell Cancer cell
Tumor-associated Tumor-associated
peptide peptide
MHC TCR TCR MHC
CD80 CD28 PD-1
Costimulatory PD-L1
signal Inhibitory
CD80 signal
Inhibitory CTLA-4
signal
Anti-PD-L1
Anti-PD-1 antibody
antibody
B
CD3ζ
Leukopheresis
First-generation CART
Transduction
with CAR retroviral
construct
Costimulatory domain 1
CD3ζ
Peripheral Second-
blood T cells generation
CART
Costimulatory domain 2
CD3ζ
Third-
generation
CART
Preconditioning
chemotherapy
Infusion of CART
Figure 1-3 Immunotherapy approaches to malignancies. (A) Immune checkpoint inhibitors. Antigen-p resenting cells and cancer
cells pres ent MHC- bound antigens to T cells. Recognition of the MHC-b ound antigen by the TCR, in addition to CD80 engagement
with CD28, leads to T-c ell activation as indicated by the “+” symbol. In contrast, engagement of CD80 with the CTLA-4 protein leads
to T-c ell inhibition (“−” symbol). PD-L 1 expression on cancer cells can associate with PD-1 on T cells, leading to inhibition in the T-c ell
effector phase. Antibodies to CTLA-4, PD-1, or PD- L1 block T-c ell inhibition, enhancing the T-c ell response to cancer cells. (B) CAR
T-c ell therapy. T cells are harvested from patients by pheresis followed by culture, transduction with a retrovirus carry ing the ge ne tic
information to encode a chimeric antigen receptor (CAR), and expansion. Patients receive a preconditioning chemotherapy regimen that
results in lymphodepletion prior to CAR T-c ell infusion. This has been demonstrated to be beneficial for enhanced in vivo CAR T- cell
expansion. The three generations of CARs are shown. First- generation CARs carry an extracellular domain that recognizes CD19 with
an intracellular domain derived from the TCR (CD3 zeta). Second-g eneration CARs include a costimulatory domain to enhance T-c ell
activation upon engagement. Third- generation CARs carry two additional costimulatory domains.
8
Downloaded from https://ashpublications.org/books/chapter-pdf/10582/ashsap7.ch01.pdf
by Manea Alqarni
on 30 May 2019